The sequencing of nucleic acid samples is an important analytical technique in modern molecular biology. The development of reliable methods for DNA sequencing has been crucial for understanding the function and control of genes and for applying many of the basic techniques of molecular biology. These methods have also become increasingly important as tools in genomic analysis and many non-research applications, such as genetic identification, forensic analysis, genetic counseling, medical diagnostics and many others. In these latter applications, both techniques providing partial sequence information, such as fingerprinting and sequence comparisons, and techniques providing full sequence determination have been employed. See, e.g., Gibbs et al., Proc. Natl. Acad. Sci. USA 86: 1919-1923 (1989); Gyllensten et al., Proc. Natl. Acad. Sci. USA 85: 7652-7656 (1988); Carrano et al., Genonmics 4: 129-136 (1989); Caetano-Annoles et al., Mol. Gen. Genet. 235: 157-165 (1992); Brenner and Livak, Proc. Natl. Acad. Sci. USA 86: 8902-8906 (1989); Green et al., PCR Methods and Applications 1: 77-90 (1991); and Versalovic et al., Nucleic Acid Res. 19: 6823-6831 (1991).
Most currently available DNA sequencing methods require the generation of a set of DNA fragments that are ordered by length according to nucleotide composition. The generation of this set of ordered fragments occurs in one of two ways: (1) chemical degradation at specific nucleotides using the Maxam-Gilbert method or (2) dideoxy nucleotide incorporation using the Sanger method. See Maxam and Gilbert, Proc Natl Acad Sci USA 74: 560-564 (1977); Sanger et al. Proc Natl Acad Sci USA 74: 5463-5467 (1977). The type and number of required steps inherently limits both the number of DNA segments that can be sequenced in parallel, and the amount of sequence that can be determined from a given site. Furthermore, both methods are prone to error due to the anomalous migration of DNA fragments in denaturing gels. Time and space limitations inherent in these eel-based methods have fueled the search for alternative methods.
In an effort to satisfy the current large-scale sequencing demands, improvements have been made to the Sanger method. For example, the use of fluorescent chain terminators simplifies detection of the nucleotides. The synthesis of longer DNA fragments and improved fragment resolution produces more sequence information from each experiment. Automated analysis of fragments in gels or capillaries has significantly reduced the labor involved in collecting and processing sequence information. See, e.g., Prober et al., Science 238: 336-341 (1987); Smith et al., Nature 321: 674-679 (1986); Luckey et al., Nucleic Acids Res 18: 4417-4421(1990); Dovichi, Electrophoresis 18: 2393-2399 (1997).
However current DNA sequencing technologies still suffer three major limitations. First, they require a large amount of identical DNA molecules, which are generally obtained either by molecular cloning or by polymerase chain reaction (PCR) amplification of DNA sequences. Current methods of detection are insensitive and thus require a minimum critical number of labeled oligonucleotides. Also, many identical copies of the oligonucleotide are needed to generate a sequence ladder. A second limitation is that current sequencing techniques depend on priming from sequence-specific oligodeoxynucleotides that must be synthesized prior to initiating the sequencing procedure. Sanger and Coulson, J. Mol. Biol. 94: 441-448 (1975). The need for multiple identical templates necessitates the synchronous priming of each copy from the same predetermined site. Third, current sequencing techniques depend on lengthy, labor-intensive electrophoresis techniques that are limited by the rate at which the fragments may be separated and are also limited by the number of bases that can be sequenced in a given experiment by the resolution obtainable on the gel.
In an effort to dispense with the need for electrophoresis techniques, a sequencing method was developed which uses chain terminators that can be uncaged, or deprotected, for further extension. See, U.S. Pat. No. 5,302,509: Metzker et al. Nucleic Acids Res. 22: 4259-4267 (1994). This method involves repetitive cycles of base incorporation, detection of incorporation, and re-activation of the chain terminator to allow the next cycle of DNA synthesis. Thus, by detecting each added base while the DNA chain is growing, the need for size-fractionation is eliminated. This method is nevertheless still highly dependent on large amounts of nucleic acid to be sequenced and the use of known sequences for priming the initiation of chain or growth. Moreover, this technique is plagued by any inefficiencies of incorporation and deprotection. Because incorporation and 3′-OH regeneration are not completely efficient. a pool of initially identical extending strands can rapidly become asynchronous and sequences cannot be resolved beyond a few limited initial additions.
Thus, a need still remains in the art for a rapid, cost effective, high throughput method for sequencing unknown nucleic acid samples that eliminates the need for amplification; prior knowledge of some of the nucleotide sequence to generate sequencing primers; and labor-intensive electrophoresis techniques.